Blood cell lysis reagent

ABSTRACT

Disclosed herein are lysis reagents for lysing red blood cells, thereby releasing an analyte, such as RNA from a host or pathogen, in a form suitable for analysis. The reagent includes at least a buffer, a detergent and one or both of a chloride containing salt and an anti-coagulant. The reagent serves to lyse blood cells, protect the released analyte from degradation in the lysate, and is compatible with subsequent steps for analysis of the analyte such as target capture, amplification, detection, or sequencing.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.16/094,070, filed on Oct. 16, 2018, which is a national stage entry ofInternational Application No. PCT/US2017/029671, filed on Apr. 26, 2017,which claims the benefit, under 35 U.S.C. § 119(e), of U.S. ProvisionalApplication No. 62/328,358, filed on Apr. 27, 2016. The entire contentsof each of the foregoing applications are hereby incorporated byreference.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII Copy, created on Dec. 22, 2020, isnamed “Dia-0023-02-Seq-Listing_ST25” and is 2,978 bytes in size.

BACKGROUND

Although there are commercial assays for detecting RNA in blood, the RNAdetected in such assays is usually present in extracellular forms, suchas HIV or HCV particles in the blood. Detection of RNA or other targetmolecules from within blood cells, and particularly from within redblood cells is more challenging. Reagents used in lysis may interferewith subsequent processing as many non-target molecules released bylysis, particularly nucleases or proteases, may degrade targetmolecules.

The intrinsic instability of RNA and presence of RNAses in whole bloodmakes isolation of RNA a difficult task. The use of high purity, intactRNA facilitates sensitive clinical diagnostic assays. Existingapproaches typically involve several sequential steps: a step to disruptthe cells, a step to denature the proteins, another step for thestabilization and protection of RNA from RNAses, and then a step forisolation of the RNA

Tetradecyltrimethylammonium oxalate (TDTMAO) is commonly used fortransport, storage and processing of blood (U.S. Pat. Nos. 6,602,718 and6,617,170). This quaternary amine is contained, for example, in thePAXgene™ Blood RNA System (BD Biosciences) and works by penetrating thecell and stabilizing intracellular target RNA. The RNA can then be laterpurified and analyzed from the components of whole blood using standardtechniques. Methods for lysing cells and inhibiting RNases usingguanidinium salts are also known (Chomczynski et al. (1987) Anal.Biochem. 162, 156-159).

SUMMARY

Provided herein is a reagent comprising a buffer and a detergent, andfurther comprising a salt, an anti-coagulant, or both. In someembodiments, the reagent comprises a buffer, a salt and a detergent. Insome embodiments, the reagent comprises a buffer, a salt and ananti-coagulant. In some embodiments, a reagent is provided comprisingone or more of a salt, a buffer, a detergent, and an anti-coagulant. Insome embodiments, a reagent is provided comprising a salt, a detergentand an anti-coagulant. In some embodiments, a reagent is providedcomprising a buffer, a salt and a detergent. In some embodiments, areagent is provided comprising a buffer, a salt, a detergent, and ananti-coagulant.

In some embodiments, the buffer is a sodium bicarbonate buffer. In someembodiments, the buffer is a TRIS(2-Amino-2-(hydroxymethyl)-1,3-propanediol) buffer. In some embodiments,the buffer is a sodium bicarbonate buffer. In some embodiments, thebuffer is a sodium phosphate buffer. In some embodiments, the buffer isa sodium bicarbonate buffer in the reagent in a concentration from about5 mM to about 30 mM, from about 10 mM to about 20 mM, about 10 mM toabout 15 mM, or from about 15 mM to about 20 mM. In some embodiments,the buffer is a TRIS buffer in the reagent at a concentration from about75 mM to about 150 mM, from about 75 mM to about 125 mM, from about 100mM to about 125 mM, or from about 90 mM to about 110 mM. In someembodiments, the buffer is a sodium phosphate (Na₃PO₄) buffer in thereagent at a concentration from about 5 mM to about 30 mM, from about 10mM to about 20 mM, about 10 mM to about 15 mM, or from about 15 mM toabout 20 mM. In some embodiments, the concentration of sodium phosphatein the reagent is from about 8 mM to about 40 mM, from about 10 mM toabout 33 mM, from about 15 mM to about 30 mM, about 30 mM, or about 15mM. In some embodiments, the concentration of sodium phosphate monobasicin the reagent is from about 8 mM to about 40 mM, from about 10 mM toabout 33 mM, from about 15 mM to about 30 mM, about 30 mM, or about 15mM. In some embodiments, the concentration of sodium phosphate dibasicin the reagent is from about 8 mM to about 40 mM, from about 10 mM toabout 33 mM, from about 15 mM to about 30 mM, about 30 mM, or about 15mM. Ranges include all whole and partial numbers therein.

In some embodiments, the anti-coagulant is one or more of EDTA((Ethylenedinitrilo)tetraacetic acid), EDTA-Na₂ (Disodiumethylenediaminetetraacetate dihydrate), EGTA(Ethylene-bis(oxyethylenenitrilo)tetraacetic acid), heparin, or citrate.In some embodiments, the anti-coagulant comprises an EDTA in the reagentat a concentration from about 0.05 mM to about 15 mM, from about 0.1 mMto about 10 mM, or from about 0.5 mM to about 5 mM. In some embodiments,the anti-coagulant is EDTA in the reagent at a concentration from about0.05 mM to about 15 mM, from about 0.1 mM to about 10 mM, or from about0.5 mM to about 5 mM. In some embodiments, the anti-coagulant isEDTA-Na₂ in the reagent at a concentration from about 0.05 mM to about15 mM, from about 0.1 mM to about 10 mM, or from about 0.5 mM to about 5mM. In some embodiments, the anti-coagulant is EGTA in the reagent at aconcentration from about 0.05 mM to about 15 mM, from about 0.1 mM toabout 10 mM, or from about 0.5 mM to about 5 mM. Ranges include allwhole and partial numbers therein.

In some embodiments, the salt comprises one or more of the followingions: a sodium ion, a potassium ion, an ammonium ion, a magnesium ion, alithium ion, and a chloride ion. In some embodiments, the salt ismagnesium chloride, ammonium chloride, potassium chloride, or sodiumchloride. In some embodiments, the salt comprises a chloride ion and oneof a magnesium ion, sodium ion or potassium ion, and the concentrationof the salt in the reagent is from about 10 mM to about 50 mM, fromabout 15 mM to about 40 mM, or from about 20 mM to about 35 mM. In someembodiments, the salt is ammonium chloride in the reagent at aconcentration from about 100 mM to about 500 mM, from about 200 mM toabout 350 mM, or from about 250 mM to about 300 mM. Ranges include allwhole and partial numbers therein.

In some embodiments, the detergent is one of lithium lauryl sulfate(LLS), nonyl phenoxypolyethoxylethanol (NP 40), sodium dodecyl sulfate(SDS), and Triton-X 100. In some embodiments, the detergent is ananionic detergent. In some embodiments, the detergent is LLS or SDS. Insome embodiments, the detergent is present in the reagent at aconcentration that is greater than about 1.5% (v/v or w/v). In someembodiments, the detergent is present in the reagent at a concentrationthat is less than about 15.5% (v/v or w/v). In some embodiments, thedetergent is present in the reagent at a concentration of from about 2%to about 15% (v/v or w/v). In some embodiments, the detergent is presentin the reagent at a concentration from about 2% to about 15% (v/v orw/v). In some embodiments, the detergent is LLS and the concentration ofLLS in the reagent is from about 2% to about 15% (w/v), from about 4% toabout 10% (w/v), or from about 5% to about 8% (w/v). In someembodiments, the detergent is LLS and is present in the reagent at about14 mM to about 50 mM. Ranges include all whole and partial numberstherein.

In some embodiments, the pH of the reagent is greater than a pH of 5.5.In some embodiments, the pH of the reagent is less than a pH of 10.5. Insome embodiments, the pH of the reagent is from about 6.0 to about 10.0.In some embodiments, the pH of the reagent is from about 6.5 to about8.0, or from about 7.0 to about 8.0, or from about 7.2 to about 7.6, orfrom about 6.7 to about 7.5, or about 6.7, or about 7.3 or about 7.5.Ranges include all whole and partial numbers therein.

In some embodiments of the reagent, the concentration of sodiumbicarbonate is 14 mM, the concentration of ammonium chloride is 250 mM,the concentration of LLS is 8% (w/v), the concentration of EDTA is fromabout 0.1 mM to about 10 mM, and the pH is 7.2-7.6. In some embodimentsof the reagent, the buffer is selected from the group consisting ofsodium bicarbonate, sodium phosphate and TRIS, the detergent is fromabout 5% to about 10% (v/v or w/v), the pH is from about 6.5 to about8.0, and the salt is selected from the group consisting of magnesiumchloride, ammonium chloride, and potassium chloride. In some embodimentsof the reagent, the buffer is selected from the group consisting ofsodium phosphate and TRIS and the salt is selected from the groupconsisting of magnesium chloride and ammonium chloride. In some aspectsof this embodiment, the concentration of anti-coagulant is about 0 mM toabout 1 mM. In some further aspects of this embodiment, the detergent isLLS at a concentration from about 6% to about 10% (w/v). In some furtheraspects of this embodiment, the buffer is TRIS at a concentration fromabout 90 mM to about 110 mM and the pH of the reagent is from about 7.2to about 7.5. In some further aspects of this embodiment, theanti-coagulant is at a concentration of about 0.1 mM to about 5 mM andis EGTA, EDTA, EDTA-Na₂ or a combination thereof.

In some embodiments, the reagent is admixed with blood cells, with redblood cells or with products derived from red blood cells. In certainembodiments, the reagent is admixed with whole blood. In someembodiments, the reagent is admixed with whole blood in a ratio of about1:1 (v/v) to about 4:1 (v/v), including all whole numbered and partialnumbered ratios there between. In some embodiments, the reagent isadmixed with whole blood in a ratio of 3:1 (v/v). In some embodiments,the whole blood is human whole blood, non-human whole blood, or amixture thereof.

Further provided herein is a method of analyzing an analyte from bloodcells comprising: (a) contacting blood cells with a reagent comprising abuffer, a salt and a detergent, the reagent being effective to lyse theblood cells and inhibit degradation of analyte released from the bloodcells; and (b) analyzing the analyte released from the blood cells.

In some methods, the target is a pathogen-derived target. In somemethods, the target is RNA.

In some methods, analyzing the target comprises a nucleic acid assay. Insome methods, analyzing the target comprises contacting the releasedtarget with a capture probe and an immobilized probe, the capture probehaving a first segment complementary to the target, and a second segmentcomplementary to the immobilized probe, wherein the target binds to thecapture probe, and wherein the bound capture probe binds to theimmobilized probe. Some methods further comprise performing atranscription mediated amplification of the target and detecting theresulting amplification product with a detection probe.

Some methods are performed without a centrifugation step to separate thereagent from the target released from the blood cells.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 sets forth the nucleic acid sequence of a non T7 primer.

SEQ ID NO:2 sets forth the nucleic acid sequence of a T7 primer.

SEQ ID NO:3 sets forth the nucleic acid sequence of an acridinium ester(AE) probe.

SEQ ID NO:4 sets forth the nucleic acid sequence of a target captureoligonucleotide (TCO) probe.

Definitions

Pathogens include viruses, bacteria, protozoa, fungi, and othermicroorganisms responsible for disease in humans and other animals.

An analyte (sometimes referred to herein as a target) can be a singletype of molecule, such as a protein or a nucleic acid, or a class ofmolecules, such as any protein or RNA from a parasite or any protein orRNA from blood cells. Multiple distinct analytes can also be analyzed,such as an RNA analyte and a protein analyte, or two distinct RNAanalytes, such as two different mRNA analytes, or an mRNA analyte and anrRNA analyte. Analytes include endogenous components of blood cells andcomponents arising as a result of pathogenic infection of infected bloodcells and are typically encoded by the infecting pathogen (i.e.,“pathogenic” or “pathogen-derived” analytes).

A lysis reagent is reagent, often provided in the form of a solution,effective for inducing lysis of blood cells in whole blood, includinglysis of red blood cells or red blood cell products such as pelleted redblood cells.

Detergents, are surface acting agents effective in solubilizinghydrophobic molecules. Generally, these are water-soluble surface-activeagents comprised of a hydrophobic portion, usually a long alkyl chain,attached to hydrophilic or water solubility enhancing functional groups.Detergents include anionic detergents, cationic detergents, zwitterionicdetergents, non-ionic detergents, and anti-foaming agents.

Anti-coagulants inhibit clotting of whole blood. Anti-coagulants includeheparins and calcium chelating agents. Heparins activate antithrombinIII, which inhibits the activity of thrombin and other proteasesinvolved in blood clotting. Calcium chelating agents, such as the EDTAs,the EGTAs and citrates, bind calcium ions required for blood clotting.

A buffer refers to a weak acid or weak base used to maintain the pH of asolution.

A nucleic acid refers to a multimeric compound comprising nucleotides oranalogs that have nitrogenous heterocyclic bases or base analogs linkedtogether to form a polymer, including conventional RNA, DNA, mixedRNA-DNA, and analogs thereof.

The nitrogenous heterocyclic bases can be referred to as nucleobases.Nucleobases can be conventional DNA or RNA bases (A, G, C, T, U), baseanalogs, (The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed.,11.sup.th ed., 1992; van Aerschott et al., 1995, Nucl. Acids Res.23(21): 4363-70), pyrimidine or purine derivatives, (Hill et al., 1998,Proc. Natl. Acad. Sci. USA 95(8):4258-63, Lin and Brown, 1992, Nucl.Acids Res. 20(19):5149-52), pyrene-functionalized LNA nucleosideanalogues (Babu & Wengel, 2001, Chem. Commun. (Camb.) 20: 2114-5;Hrdlicka et al., 2005, J. Am. Chem. Soc. 127(38): 13293-9), andhydrophobic nucleobases that form duplex DNA without hydrogen bonding(Berger et al., 2000, Nucl. Acids Res. 28(15): 2911-4). Many derivatizedand modified nucleobases or analogues are commercially available (e.g.,Glen Research, Sterling, Va.).

A nucleobase unit attached to a sugar, can be referred to as anucleobase unit, or monomer. Sugar moieties of a nucleic acid can beribose, deoxyribose, or similar compounds, e.g., with 2′ methoxy or 2′halide substitutions. Nucleotides and nucleosides are examples ofnucleobase units.

The nucleobase units can be joined by a variety of linkages orconformations, including phosphodiester, phosphorothioate ormethylphosphonate linkages, peptide-nucleic acid linkages (PNA; Nielsenet al., 1994, Bioconj. Chem. 5(1): 3-7; PCT No. WO 95/32305), and alocked nucleic acid (LNA) conformation in which nucleotide monomers witha bicyclic furanose unit are locked in an RNA mimicking sugarconformation (Vester et al., 2004, Biochemistry 43(42):13233-41;Hakansson & Wengel, 2001, Bioorg. Med. Chem. Lett. 11 (7):935-8), orcombinations of such linkages in a nucleic acid strand. Nucleic acidsmay include one or more “abasic” residues, i.e., the backbone includesno nitrogenous base for one or more positions (U.S. Pat. No. 5,585,481).

A nucleic acid may include only conventional RNA or DNA sugars, basesand linkages, or may include both conventional components andsubstitutions (e.g., conventional RNA bases with 2′-O-methyl linkages,or a mixture of conventional bases and analogs). Inclusion of PNA,2′-methoxy or 2′-fluoro substituted RNA, or structures that affect theoverall charge, charge density, or steric associations of ahybridization complex, including oligomers that contain charged linkages(e.g., phosphorothioates) or neutral groups (e.g., methylphosphonates)may affect the stability of duplexes formed by nucleic acids.

An oligomer may contain a “random polymer” sequence that refers to apopulation of oligomers that are substantially the same in overalllength and other characteristics, but in which at least a portion of theoligomer is synthesized by random incorporation of different bases for aspecified length, e.g., a random assortment of all four standard bases(A, T, G, and C) in a DNA oligomer, or a random assortment of a fewbases (U or T and G) in a defined portion of a larger oligomer. Theresulting oligomer is actually a population of oligomers whose finitenumber of members is determined by the length and number of bases makingup the random portion (e.g., 2⁶ oligomers in a population of oligomersthat contains a 6-nt random sequence synthesized by using 2 differentbases).

Complementarity of nucleic acids means that a nucleotide sequence in onestrand of nucleic acid, due to orientation of its nucleobase groups,hydrogen bonds to another sequence on an opposing nucleic acid strand.The complementary bases typically are, in DNA, A with T and C with G,and, in RNA, C with G, and U with A. Complementarity can be perfect(i.e., exact) or substantial/sufficient. Perfect complementarity betweentwo nucleic acids means that the two nucleic acids can form a duplex inwhich every base in the duplex is bonded to a complementary base byWatson-Crick pairing. “Substantial” or “sufficient” complementary meansthat a sequence in one strand is not completely and/or perfectlycomplementary to a sequence in an opposing strand, but that sufficientbonding occurs between bases on the two strands to form a stable hybridcomplex in set of hybridization conditions (e.g., salt concentration andtemperature). Such conditions can be predicted by using the sequencesand standard mathematical calculations to predict the Tm of hybridizedstrands, or by empirical determination of Tm by using routine methods.Tm refers to the temperature at which a population of hybridizationcomplexes formed between two nucleic acid strands are 50% denatured. Ata temperature below the Tm, formation of a hybridization complex isfavored, whereas at a temperature above the Tm, melting or separation ofthe strands in the hybridization complex is favored. Tm may be estimatedfor a nucleic acid having a known G+C content in an aqueous 1 M NaClsolution by using, e.g., Tm=81.5+0.41 (% G+C), although other known Tmcomputations take into account nucleic acid structural characteristics.

“Separating” or “isolating” or “purifying” refers to removing one ormore components from a complex mixture, such as a sample. Preferably, aseparating, isolating or purifying step removes at least 70%, preferablyat least 90%, and more preferably at least 95% w/w of the nucleic acidanalytes from other sample components. A separating, isolating orpurifying step may optionally include additional washing steps to removenon-analyte sample components.

“Release” of a capture hybrid refers to separating one or morecomponents of a capture hybrid from each other, such as separating anucleic acid analyte from a capture probe, and/or a capture probe froman immobilized probe. Release of the nucleic acid strand separates theanalyte from other components of a capture hybrid and makes the analyteavailable for binding to a detection probe. Other components of thecapture hybrid may remain bound, e.g., the capture probe strand to theimmobilized probe on a capture support, without affecting analytedetection.

A “label” refers to a molecular moiety that is detectable or produces adetectable response or signal directly or indirectly, e.g., bycatalyzing a reaction that produces a detectable signal. Labels includeluminescent moieties (such as fluorescent, bioluminescent, orchemiluminescent compounds), radioisotopes, members of specific bindingpairs (e.g., biotin and avidin), enzyme or enzyme substrate, reactivegroups, or chromophores, such as a dye or particle that results indetectable color.

A capture probe includes a first segment including atarget-complementary region of sequence and a second segment forattaching the capture probe, or a hybridization complex that includesthe capture probe, to an immobilized probe. The first segment can beconfigured to substantially complementary to a specific nucleic acidanalyte sequence (or target sequence) so that a first segment and atarget nucleic acid can hybridize to form a stable duplex (i.e., havinga detectable melting point) under hybridizing conditions, such asdescribed in the Examples. Alternatively, the first segment can beconfigured to nonspecifically bind to nucleic acid sequences in a sampleunder hybridizing conditions (see WO 2008/016988). The second segmentincludes a region of sequence that is complementary to a sequence of animmobilized probe. Preferably, a chimeric capture probe includes anucleic acid homopolymer (e.g., poly-A or poly-T) that is covalentlyattached to the target-complementary region of the capture probe andthat hybridizes under appropriate conditions to a complementaryhomopolymer of the immobilized probe (e.g., poly-T or poly-A,respectively) as previously described (U.S. Pat. No. 6,110,678 toWeisburg et al.). Capture probes may further comprise a third segmentthat acts as a closing sequence to inactivate unbound target captureprobes in a capture reaction. This third segment can flank the firstsegment opposite the second segment (e.g., capture sequence:targethybridizing sequence:closing sequence) or it can flank the secondsegment opposite the first segment (e.g., closing sequence:capturesequence:target hybridizing sequence). See WO 2006/007567 and US2009-0286249.

An immobilized probe includes a nucleic acid joined directly orindirectly to a support. The nucleic acid is complementary to a nucleicacid in the capture probe, although may or may not be the same length(number of nucleobase units) as the in the capture probe. The nucleicacid in the immobilized probe preferably contains at least sixcontiguous nucleobase units and can contain for example 10-45 or 10-40or 10-30 or 10-25 or 15-25, inclusively, L-nucleobase units. The nucleicacid is preferably a homopolymer, and more preferably a homopolymer ofadenine or thymine. A preferred form of immobilized probe is or includesa homopolymer of 14 thymine residues for use in combination with acapture probe including a second segment with a homopolymer of adenineresidues. The nucleic acid moiety of an immobilized probe is typicallyprovided in single-stranded form, or if not, is denatured tosingle-stranded form before or during use.

Any of a variety of materials may be used as a support for theimmobilized probes, e.g., matrices or particles made of nitrocellulose,nylon, glass, polyacrylate, mixed polymers, polystyrene, silanepolypropylene, and magnetically attractable materials. Monodispersemagnetic spheres are a preferred support because they are relativelyuniform in size and readily retrieved from solution by applying amagnetic force to the reaction container, preferably in an automatedsystem. An immobilized probe may be linked directly to the capturesupport, e.g., by using any of a variety of covalent linkages,chelation, or ionic interaction, or may be linked indirectly via one ormore linkers joined to the support. The linker can include one or morenucleotides not intended to hybridize to the capture probe but to act asa spacer between the nucleic acid of the immobilized probe and itssupport.

A “detection probe” is a nucleic acid or other molecule that bindsspecifically to a target sequence and which binding results, directly orindirectly, in a detectable signal to indicate the presence of thetarget sequence. A detection probe need not be labeled to produce adetectable signal, e.g., an electrical impulse resulting from bindingthe probe to its target sequence may be the detectable signal. A“labeled probe” is a probe that contains or is linked, directly orindirectly, to a label (e.g., Sambrook et al., Molecular Cloning, ALaboratory Manual, 2nd ed., Chapt. 10; U.S. Pat. No. 6,361,945, Beckeret al.; U.S. Pat. No. 5,658,737, Nelson et al.; U.S. Pat. No. 5,656,207,Woodhead et al.; U.S. Pat. No. 5,547,842, Hogan et al.; U.S. Pat. No.5,283,174, Arnold et al.; U.S. Pat. No. 4,581,333, Kourilsky et al.;U.S. Pat. No. 5,731,148, Becker et al.). For example, detection probesmay include a non-nucleotide linker and a chemiluminescent labelattached to the linker (U.S. Pat. Nos. 5,185,439, 5,585,481 and5,639,604, Arnold et al.). Examples of detection probes includeoligonucleotides of about 5 to 50 nucleotides in length having anattached label that is detected in a homogeneous reaction, e.g., onethat uses differential hydrolysis of a label on a bound or unboundprobe.

Detection probes can have a nucleotide sequence that is of the same oropposite sense as a target sequence depending on the format of theassay. Detection probes can hybridize to the same or different segmentof a target sequence as a capture probe. Some detection probes have anattached chemiluminescent marker, e.g., an acridinium ester (AE)compound (U.S. Pat. Nos. 5,185,439, 5,639,604, 5,585,481, and5,656,744). In some detection probes, an acridinium ester label isattached to a central region of the probe near a region of A and T basepairs by using a non-nucleotide linker (U.S. Pat. Nos. 5,585,481 and5,656,744, Arnold, et al.) which restricts the amines of the nucleotidebases on both sides of the AE and provides a site for intercalation.Alternatively, an AE label may be attached to the 3′ or 5′ terminus ofthe detection probe which is used in conjunction with a second oligomerthat hybridizes adjacent to the detection probe on the target nucleicacid to restrict the effects of nearby amine contributed by the targetnucleic acid. In some detection probes, an AE label at or near the siteof a mismatch with a related non-target polynucleotide sequence, topermit discrimination between the related sequence and the targetsequence that may differ by only one nucleotide because the area of theduplex around the mismatch site is sufficiently destabilized to renderthe AE on the probe hybridized to the related non-target sequencesusceptible to hydrolysis degradation. HIV-1 and HCV may be detectedusing a modified form of the commercial PROCLEIX® ULTRIO HIV-1/HCV/HBVAssay from Gen-Probe. The modification involves replacing the D-polyAand D-polyT sequences in capture and immobilized probes with L-poly Aand L-poly-T, respectively.

“Hybridization condition” refers to the cumulative environment in whichone nucleic acid strand bonds to a second nucleic acid strand bycomplementary strand interactions and hydrogen bonding to produce ahybridization complex. Such conditions include the chemical componentsand their concentrations (e.g., salts, chelating agents, formamide) ofan aqueous or organic solution containing the nucleic acids, and thetemperature of the mixture. Other factors, such as the length ofincubation time or reaction chamber dimensions may contribute to theenvironment (e.g., Sambrook et al., Molecular Cloning, A LaboratoryManual, 2.sup.nd ed., pp. 1.90-1.91, 9.47-9.51, 11.47-11.57 (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989)).

Specific binding of a target capture oligomer to a target nucleic acidor target nucleic acids means binding between a single defined sequencein the first segment of a target capture oligomer and an exactly orsubstantially complementary segment on target nucleic acid(s) to form astable duplex. Such binding is detectably stronger (higher signal ormelting temperature) than binding to other nucleic acids in the samplelacking a segment exactly or substantially complementary to the singledefined target capture oligomer sequence. Non-specific binding of atarget capture oligomer to target nucleic acids means that the targetcapture oligomer can bind to a population of target sequences that donot share a segment having exact or substantial complementarity to asingle defined target capture oligomer sequence. Such can be achieved byfor example using a randomized sequence in the first segment of thecapture probe.

Lack of binding between nucleic acids can be manifested by bindingindistinguishable from nonspecific binding occurring between a randomlyselected pair of nucleic acids lacking substantial complementarity butof the same lengths as the nucleic acids in question.

“Release” of a capture hybrid refers to separating one or morecomponents of a capture hybrid from each other, such as separating atarget nucleic acid from a capture probe, and/or a target captureoligomer from an immobilized probe. Release of the target nucleic acidstrand separates the analyte from other components of a capture hybridand makes the analyte available for binding to a detection probe. Othercomponents of the capture hybrid may remain bound, e.g., the targetcapture oligomer strand to the immobilized probe on a capture support,without affecting analyte detection.

“Sensitivity” is the proportion of true positives correctly identifiedas such (e.g. the percentage of infected patients correctly identifiedas having the infection). Specificity measures the proportion of truenegatives which are correctly identified (e.g. the percentage ofuninfected patients who are correctly identified as not having theinfection.)

Reference to a range of values also includes integers within the rangeand sub-ranges defined by integers in the range. Reference to anynumerical value or range of numerical values should be understand asencompassing any such variation as is inherent in measuring that valueother typical conditions of use.

DETAILED DESCRIPTION I. General

Provided herein is a lysis reagent for lysing blood cells, therebyreleasing RNA or other analyte in a form suitable for analysis.Preferably, the lysis reagent lysis blood cells, including red bloodcells, thereby releasing RNA or other analyte in a form suitable foranalysis. In one aspect, the lysis reagent lysis a sample comprising,consisting of, or consisting essentially of blood cells, therebyreleasing RNA or other analyte in a form suitable for analysis. Thelysis reagent comprises at least a buffer, a salt and a detergent. Thereagent serves to lyse blood cells, protect a released analyte fromdegradation in the lysate, and is compatible with subsequent steps foranalysis of the analyte, such as target capture, amplification,detection, and/or sequencing. The lysis reagent is amenable for analysisof an analyte from a pathogen or a host. Analytes are preferably nucleicacid analytes from a pathogen or from a host. More preferably, analytesare RNA analytes from a pathogen or from a host. The lysis reagent isparticularly amenable for analysis of nucleic acids from pathogensinfecting blood cells, including, but not limited to: hepatitis viruses,human immunodeficiency viruses, dengue viruses, west nile viruses,flaviviruses, such as zika virus, and parasitic organisms such asBabesia and Plasmodium species.

The disclosed lysis reagent results in part from identifyingdeficiencies with various known lysis agents for preparing and analyzingpathogen-derived RNA from red blood cells; though the lysis reagent canbe used for preparing a number of components from blood cells. Knownlysis agents were found to be incompatible with reagents and methods foranalyzing pathogen-derived RNA, causing cell clumping, the appearance ofprecipitate, and the loss of magnetic beads when lysed samples wereadded to capture reagents. By contrast, the present lysis reagent wascompatible with these methods, allowing for the lysis of blood cells inwhole blood samples and the sensitive detection of the releasedpathogen-derived RNA following target capture and transcription mediatedamplification. The present lysis reagent also inhibited degradation ofthe pathogen-derived RNA by nucleases and proteases following lysis anddemonstrated reproducibility between samples.

II. Lysis Reagents

The present lysis reagent comprises at least a buffer, a detergent andone or both of a salt and an anti-coagulant. Buffers are present in thelysis reagent at a concentration from about 5 mM to about 150 mM. Sodiumbicarbonate is one example of a suitable buffer (NaHCO₃). Sodiumbicarbonate buffer can be present in the reagent at a concentration of,for example, from about 5 mM to about 30 mM, from about 10 mM to about20 mM, from about 10 mM to about 15 mM, from about 15 mM to about 20 mM,from about 12 mM to about 16 mM or at about 14 mM. TRIS buffer can bepresent in the reagent at a concentration of, for example from about 75mM to about 150 mM, from about 75 mM to about 125 mM, from about 100 mMto about 125 mM, from about 90 mM to about 110 mM, or at about 100 mM.Sodium phosphate buffer can be present in the reagent at a concentrationof, for example, from about 5 mM to about 40 mM, from about 10 mM toabout 33 mM, from about 15 mM to about 30 mM, about 30 mM, or about 15mM. Sodium phosphate monobasic buffer can be present in the reagent at aconcentration of, for example, from about 8 mM to about 40 mM, fromabout 10 mM to about 33 mM, from about 15 mM to about 30 mM, about 30mM, or about 15 mM. Sodium phosphate dibasic buffer can be present inthe reagent at a concentration of, for example, from about 8 mM to about40 mM, from about 10 mM to about 33 mM, from about 15 mM to about 30 mM,about 30 mM, or about 15 mM.

The pH of the reagent can be, for example, from about 6.0 to about 10.0,from about 6.5 to about 9.0, from about 7.0 to about 8.0, from about 7.2to about 7.6, about 7.5, about 7.3, or about 6.7. Ranges include allwhole and partial numbers therein.

Detergents can act as both a lysing agent and as an inhibitor of analytedegradation following the lysis of blood cells. Detergents areparticularly useful for inhibiting the degradation of nucleic acids.Exemplary detergents include Triton X-100, nonylphenoxypolyethoxylethanol (NP-40), lithium lauryl sulfate (LLS) orsodium dodecyl sulfate (SDS). LLS is preferred. By way of example, aconcentration range of detergent in the lysis reagent includes fromabout 2% to about 15% (v/v or w/v), from about 4% to about 10% (v/v orw/v), from about 5% to about 8% (v/v or w/v), about 6% (v/v or w/v),about 8% (v/v or w/v), or about 10% (v/v or w/v).

Salts, if present in the lysis reagent, are at a concentration fromabout 10 mM to about 1,000 mM. Exemplary concentration ranges forammonium chloride in the reagent include from about 100 mM to about 1000mM, from about 100 mM to about 800 mM, from about 100 mM to about 500mM, from about 150 mM to about 300 mM, from about 200 mM to about 300mM, from about 240 to about 260 mM, or about 250 mM. Exemplaryconcentration ranges for magnesium chloride in the reagent include fromabout 10 mM to about 300 mM, from about 15 mM to about 200 mM, fromabout 20 mM to about 100 mM, from about 25 mM to about 50 mM, from about28 mM to about 40 mM, from about 30 mM to about 35 mM, about 33 mM, orabout 30 mM. Exemplary concentration ranges for potassium chloride inthe reagent include from about 10 mM to about 300 mM, from about 15 mMto about 200 mM, from about 20 mM to about 100 mM, from about 25 mM toabout 50 mM, from about 28 mM to about 40 mM, from about 30 mM to about35 mM, about 33 mM, or about 30 mM.

The anti-coagulant, if present in the lysis reagent, is at aconcentration sufficient to inhibit clotting of the sample (e.g., wholeblood or red blood cells). By inhibiting clotting, the anti-coagulanteliminates the need to centrifuge samples during the method to isolatered blood cells. Exemplary anti-coagulants include EDTA EDTA-Na₂, EGTA,heparin, or citrate. Exemplary concentrations of EDTA in the lysisreagent include from about 0.05 mM to about 15 mM, from about 0.1 mM toabout 10 mM, from about 0.5 mM to about 5 mM, about 10 mM, about 2.5 mMor about 0.1 mM. Exemplary concentrations of EDTA-Na₂ in the lysisreagent include from about 0.05 mM to about 15 mM, from about 0.1 mM toabout 10 mM, from about 0.5 mM to about 5 mM, about 10 mM, about 2.5 mM,or about 0.1 mM. Exemplary concentrations of EGTA in the lysis reagentinclude from about 0.05 mM to about 15 mM, from about 0.1 mM to about 10mM, from about 0.5 mM to about 5 mM, about 7.5 mM, about 3 mM or about 1mM.

A preferred lysis reagent includes sodium bicarbonate, ammoniumchloride, LLS, and EDTA in a powder form or in a solvent, such as water,at any of the concentrations indicated above. Preferably sodiumbicarbonate is at a concentration of 12 mM to 16 mM or, more preferablyat 14 mM; ammonium chloride is at a concentration of 100 mM to 500 mMor, more preferably 250 mM, LLS is at a concentration of 4% to 15% or,more preferably 8% (w/v), EDTA is at a concentration of 0.01 mM to 10 mMor, more preferably, 0.1 mM or 10 mM; and the pH of the reagent is 7.2to 7.6 or, more preferably, 7.3. Optionally, the lysis reagent consistsessentially of sodium bicarbonate, ammonium chloride, LLS, EDTA, andwater.

A preferred lysis reagent includes sodium phosphate, LLS, EDTA-Na₂, andEGTA in a powdered form or in a solvent, such as water, at any of theconcentrations indicated above. Preferably, the sodium phosphate bufferis at a concentration of from about 5 mM to about 30 mM or, morepreferably, 30 mM or 15 mM; the LLS is at a concentration of 4% to 15%or, more preferably 10% (w/v); the EDTA-Na₂ is at a concentration of 0.5mM to 5 mM or, more preferably, 1 mM; and the EGTA is at a concentrationof 0.5 mM to 5 mM or, more preferably, 1 mM; and the pH of the reagentis 6.0 to 8.0 or, more preferably, 6.7. Preferably, the sodium phosphatebuffer comprises one or both of sodium phosphate monobasic and sodiumphosphate dibasic. Preferably, sodium phosphate buffer comprises sodiumphosphate monobasic at a concentration of from about 5 mM to about 30 mMor, more preferably, 30 mM or 15 mM. Preferably, sodium phosphate buffercomprises sodium phosphate dibasic at a concentration of from about 5 mMto about 30 mM or, more preferably, 30 mM or 15 mM. Preferably, sodiumphosphate buffer comprises sodium phosphate monobasic at a concentrationof from about 5 mM to about 30 mM or, more preferably, 30 mM or 15 mMand comprises sodium phosphate dibasic at a concentration of from about5 mM to about 30 mM or, more preferably, 30 mM or 15 mM. Optionally, thelysis reagent consists essentially of sodium phosphate, detergent,EDTA-Na₂, EGTA, and water.

A preferred lysis reagent includes TRIS, magnesium chloride, and LLS ina powdered form or in a solvent, such as water, at any of theconcentrations indicated above. Preferably TRIS is at a concentration of75 mM to 150 mM or, more preferably 100 mM; magnesium chloride is at aconcentration of 10 mM to 50 mM or, more preferably, 30 mM; the LLS isat a concentration of 4% to 15% or, more preferably 6% (w/v); and the pHof the reagent is 7.0 to 8.0 or, more preferably, 7.5. Optionally, thelysis reagent consists essentially of TRIS, magnesium chloride, and LLS,and water. Optionally, the lysis reagent contains an anti-foaming agent.

The lysis reagent can be provided as a kit also including capture probe,immobilized probe, solid support, detection probe and or primers forperforming an assay on an analyte to be isolated from blood cells,including any of the analytes described below. Such a kit can includeinstructions for using the lysis reagent and/or performing an assay onan analyte isolated from blood cells. Reaction mixtures can be preparedfrom the kits, including blood cell lysis reaction mixtures, targetcapture reaction mixtures, nucleic acid amplification reaction mixtures,nucleic acid detection reaction mixtures, and combinations thereof. Somereaction mixtures contain the lysis reagent disclosed herein.

III. Use of Lysis Reagents

Whole blood can be obtained from a number of sources, including directlyfrom whole blood donors or from blood banking facilities. Red bloodcells can be obtained from any available source, such as whole blood orany fraction thereof that includes red blood cells, such as pelleted redblood cells. Whole blood can be human whole blood, non-human wholeblood, or a combination thereof.

The lysis reagent can be admixed with blood cells for a time sufficientto induce cell lysis and cause release of molecules of desiredanalyte(s) from cells. Exemplary times for maintaining blood cellsadmixed with lysis reagent include 1-30 minutes, 2-15 minutes, 3-10minutes, 4-6 minutes, or 5 minutes. Preferably, the time is no more than30, 15, 10 or 5 minutes. Preferably the mixture lacks visible particlesafter lysis. Ranges include all whole and partial numbers therein.

The temperature of incubation of the lysis reagent with blood cells canvary. The temperature is preferably chosen to maximize extent and rateof lysis and to minimize degradation of analyte(s) or prevent inhibitionof subsequent processing. Exemplary temperature ranges include 0-50° C.,5-45° C., 10-40° C., 15-37° C., 20-30° C., 22-27° C., or 25° C. Ambienttemperature is suitable. Lysis of blood cells should release asufficient amount of analyte molecules to be detectable by the methodsdescribed herein. Preferably lysis results in at least 50%, 60%, 70%,80%, 90%, or 100% lysis of blood cells in a sample being lysed. Rangesinclude all whole and partial numbers therein.

The ratio at which whole blood is combined with lysis reagent can affectthe extent and rate of cell lysis and protection of analyte moleculesfrom degradation after release from lysed cells. Exemplary ratios inwhich whole blood is admixed with the lysis reagent include ratios of1:1, 1:2, 1:3, 1:4, 1:5, 1:10, or in a range of ratios between 1:1 and1:10 (v/v; whole blood:reagent). A preferred ratio is whole bloodadmixed with the lysis reagent at a ratio of about 1:2 to about 1:4, or1:2, or 1:3, or 1:4 (v/v). When the sample comprises red blood cellsisolated from whole blood, such as pelleted red blood cells, the redblood cells can be admixed with the lysis reagent at exemplary ratios of1:1, 1:2, 1:3, 1:4, 1:5, 1:10, or in a range of ratios between 1:1 and1:10 (v/v; red blood cells:reagent). Ranges include all whole andpartial numbers therein.

IV. Analytes

Analytes released from blood cells, including from red blood cells, bythe present reagent can be analytes from a pathogen or analytes from ahost. Analytes released from blood cells, including from red bloodcells, by the present lysis reagent can include nucleic acids (e.g., DNAor RNA), whole particles, proteins, and antibodies. Analytes arepreferably nucleic acid analytes from a pathogen or from a host. Morepreferably, analytes are RNA analytes from a pathogen or from a host.Various types of RNA analytes can be detected. The RNA analytes can beribosomal RNA (rRNA), messenger RNA (mRNA), or heterogeneous nuclear RNA(hnRNA). A preferred analyte for pathogen-derived analytes is ribosomalRNA, particularly 18S rRNA, 5S rRNA, 5.8S rRNA, or 28S rRNA.

Exemplary pathogens include those that can be detected from blood cells,including, but not limited to, hepatitis viruses, human immunodeficiencyviruses, dengue viruses, west nile viruses, flaviviruses, such as zikavirus, and parasitic organisms. Exemplary parasitic organisms includeparasites from the genus Babesia, Plasmodium, Trypanosoma, Leishmania,Anaplasma, or Toxoplasma. Organisms of the genus Babesia that causedisease in humans can be Babesia microti, Babesia divergens, or Babesiaduncani. Organisms of the genus Plasmodium can be Plasmodium falciparum,Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, or Plasmodiumknowlesi.

V. Assays

Analyte molecules released from lysis of blood cells are subject toanalysis.

Analyte molecules may or may not be separated from the lysis reagent (bycentrifugation or otherwise) before analysis. Omission of a separationstep can facilitate efficient work flow in performing the assay. Thetype of assay depends on the analyte.

A. Nucleic Acids

Analysis of nucleic acid analytes often involves steps of capture,amplification and detection. Alternatively, amplification and detectionmethods can be performed without prior target capture. Preferablyamplification, and detection and target capture (if performed) occurwithout separation of analyte molecules from the lysis reagent. Thus,the entire process can be performed in a single vessel.

1. Target Capture Assay

An exemplary target capture assay can be performed as follows using oneor more capture probes, an immobilized probe, a sample, and a suitablemedium to permit hybridization of the target capture oligomer to thetarget nucleic acid and of target capture oligomer to the immobilizedprobe. The sample can be heated (e.g., from 65° C. to 95° C.) beforeperforming the assay to denature any nucleic acids in double-strandedform. The components can be mixed in any order. For example the targetcapture oligomer can be added to the sample and hybridized with thetarget nucleic acid in the sample before adding the immobilized probe.However, for an automated assay, it is preferable to minimize the numberof adding steps by supplying the target capture oligomer and immobilizedprobe at the same or substantially the same time. In this case, theorder of hybridization can be controlled by performing a firsthybridization under conditions in which a duplex can form between thetarget capture oligomer and the target nucleic acid but which exceedsthe melting temperature of the duplex that would form between first andsecond stem segments of the capture probe and between the target captureoligomer and immobilized probe, and then performing a secondhybridization under conditions of reduced stringency, preferably belowthe melting temperature of the duplexes formed between the first andsecond stem segments and between the target capture oligomer and theimmobilized probe. Stringency can be reduced by lowering the temperatureof the assay mix. At the higher temperature, the target binding siteduplexes with the target nucleic acid. At the lower temperature, thefirst and second stem segments of capture probes not bound to the targetnucleic acid duplex with one another and the first stem segment ofcapture probes bound to the target nucleic acid duplexes with theimmobilized probe. For example, the higher stringency hybridization canbe performed at or around 60° C. and the lower stringency hybridizationby allowing cooling to room temperature or 25° C. Stringency can also bereduced by reducing salt concentration or adding or increasingconcentration of a chaotropic solvent. In some methods, all steps (withthe possible exception of an initial denaturation step at highertemperature to denature double stranded target) can be performedisothermally.

Following formation of the target nucleic acid: capture probe,immobilized probe hybrid (the capture hybrid complex) is separated awayfrom other sample components by physically separating the capturesupport using any of a variety of known methods, e.g., centrifugation,filtration, or magnetic attraction of a magnetic capture support. Theseparation is preferably performed at a temperature below the meltingtemperature of stem-loop structures formed by target capture oligomersso that empty target capture oligomers have no opportunity to denatureand thus bind to the capture probe. In some methods, the separation isperformed at a temperature less than but within 10° C. of the meltingtemperature of the stem-loop structure (e.g., at 60° C.) to maintainstringency of hybridization conditions and consequent ability todistinguished matched and unmatched target nucleic acids.

To further facilitate isolation of the target nucleic acid from othersample components that adhere non-specifically to any portion of thecapture hybrid, the capture hybrid may be washed one or more times todilute and remove other sample components. Washing may be accomplishedby dissociating the capture hybrid into its individual components in anappropriate aqueous solution (e.g., a solution containing Tris and EDTA.See e.g., U.S. Pat. No. 6,110,678) and appropriate conditions (e.g.,temperature above the T_(m) of the components) and then readjusting theconditions to permit reformation of the capture hybrid. However, forease of handling and minimization of steps, washing preferably rinsesthe intact capture hybrid attached to the capture support in a solutionby using conditions that maintain the capture hybrid. Preferably,capture of the target nucleic acid with washing if performed, isolatesat least 70%, preferably at least 90%, and more preferably about 95% ofthe target nucleic acids away from other sample components. Isolatednucleic acids can be used for a number of downstream processes, such asnucleic acid amplification.

A target capture assay may also be performed as part of a real-time,biphasic, target capture and amplification method. In such a method, 500μL of sample and 400 μL of target capture reagent (TCR) are added toreaction tubes. The TCR contains magnetic particles, components to lyseorganisms present in the sample, capture oligos, a T7 initiationpromoter, and an internal calibrator. Fluid in the reaction tubes ismixed for a specific time and speed to ensure the mixture ishomogeneous. Reaction tubes are then transferred to a transitionincubator at 43.7° C. to preheat the fluid in the reaction tubes.Reaction tubes are then transferred to an anneal incubator set at 64° C.During incubation at 64° C., any organisms present in the sample thatwere not previously disrupted by the lysis reagent are disrupted,causing release of the analyte. Reaction tubes are then moved to atransition incubator to start a cool down process, and are furthercooled in a chiller ramp (17° C. to 19° C.), leading to binding of theT7 initiation promoter and capture of both the analyte and the internalcalibrator to the magnetic particles via the capture oligos. Thereaction tubes are moved to a magnetic parking station where they aresubjected to magnets which pull the magnetic particles to the sides ofthe tubes prior to entering a wash station. In the wash station,potential interfering substances are removed from the reaction bywashing the magnetic particles.

2. Amplification

A nucleic acid analyte can be amplified using methods such as isothermalamplification reactions (e.g., transcription mediated amplification(TMA), nucleic acid sequence based amplification (NASBA), loop mediatedisothermal amplification, polymerase spiral reaction (PSR) (Liu, W. etal. Polymerase Spiral Reaction (PSR): A novel isothermal nucleic acidamplification method. Sci. Rep. 5, 12723; (2015)), ligase chainreaction, and other isothermal amplification methods), or temperaturecycling amplification reactions (e.g., polymerase chain reaction (PCR),quantitative PCR (qPCT), real time PCR (rt-PCT), or other temperaturecycling amplification methods), or other amplification methods.Detection of the amplified RNA analyte products can be performed duringamplification (real-time) or following amplification (end-point).

i. Transcription Mediated Amplification

TMA has been previously described (e.g., U.S. Pat. Nos. 5,399,491,5,554,516, 5,824,518 and 7,833,716; and also e.g., F. Gonzales and S.McDonough. Applications of Transcription-Mediated Amplification toQuantification of Gene Sequences. Gene Amplification. 1998 Ed. FrancoisFerre, Birkhauser, Boston. PP. 189-204). In TMA, a target nucleic acidthat contains the sequence to be amplified is provided as singlestranded nucleic acid (e.g., ssRNA or ssDNA). Any conventional method ofconverting a double stranded nucleic acid (e.g., dsDNA) to asingle-stranded nucleic acid may be used. A promoter primer bindsspecifically to the analyte nucleic acid at its target sequence and areverse transcriptase (RT) extends the 3′ end of the promoter primerusing the target strand as a template to create a cDNA copy, resultingin a RNA:cDNA duplex. RNase activity (e.g., RNase H of RT enzyme)digests the RNA of the RNA:cDNA duplex and a second primer bindsspecifically to its target sequence in the cDNA, downstream from thepromoter-primer end. Then RT synthesizes a new DNA strand by extendingthe 3′ end of the second primer using the cDNA as a template to create adsDNA that contains a functional promoter sequence. RNA polymerasespecific for the functional promoter initiates transcription to produceabout 100 to 1000 RNA transcripts (amplified copies or amplicons)complementary to the initial target strand. The second primer bindsspecifically to its target sequence in each amplicon and RT creates acDNA from the amplicon RNA template to produce a RNA:cDNA duplex. RNasedigests the amplicon RNA from the RNA:cDNA duplex and thetarget-specific sequence of the promoter primer binds to itscomplementary sequence in the newly synthesized DNA and RT extends the3′ end of the promoter primer as well as the 3′ end of the cDNA tocreate a dsDNA that contains a functional promoter to which the RNApolymerase binds and transcribes additional amplicons that arecomplementary to the target strand. Autocatalytic cycles that use thesesteps repeatedly during the reaction produce about a billion-foldamplification of the initial target sequence. Optionally, amplicons maybe detected during amplification (real-time detection) or at an endpoint of the reaction (end-point detection) by using a probe that bindsspecifically to a sequence contained in the amplicons. Detection of asignal resulting from the bound probes indicates the presence of thetarget nucleic acid in the sample.

TMA may also be performed as part of a real-time, biphasic, targetcapture and amplification method. In such a method, TMA can be performedby adding amplification reagent (50 μL/test) to reaction tubescontaining captured analyte molecules and mixing in an amplificationload station. The amplification reagent contains oligos and componentsnecessary to build nucleic acids. The reaction tubes are moved to atransition incubator at 43.7° C. to increase the temperature of theliquid in the reaction tubes, which are then moved back to theamplification load station where enzyme (25 μL/test) is added. Reactiontubes are moved to the amplification incubator set at 42.7° C. andremain in the incubator for five minutes, during which the first roundsof amplification are initiated. Reaction tubes are moved back to theamplification load station where promoter reagent (25 μL/test) is added.Reaction tubes are moved back to the amplification incubator for furtherrounds of analyte amplification. The promoter reagent contains oligosand torches. The torches are complementary to the analyte or internalcalibrator and fluoresce when bound, generating signal in real-time. Thesignals for the target and internal calibrator preferably have differentwavelengths and can be distinguished.

ii. Polymerase Chain Reaction

Alternatively, PCR amplification (e.g., reverse transcriptase orreal-time PCR) can be used for amplification. PCR can be performed withor without prior release of the target nucleic acid from the capturecomplex. The PCR reaction can be performed in the same vessel (e.g., amicrofuge tube) as the capture step. The PCR reaction involvesthermocycling between a high temperature of about 95° C. (e.g., 90-99°C.) for dissociation and a low temperature of about 60° C. e.g., 40-75,or 50-70 or 55-64° C.) for annealing. Typically, the number of completethermocycles is at least 10, 20, 30 or 40. PCR amplification isperformed using one or more primer pairs. A primer pair used for PCRamplification includes two primers complementary to opposite strands ofa target nucleic acid flanking the region desired to be sequenced. Forsequencing most of a viral genome (e.g., more than 50, 75 or 99%), theprimers are preferably located close to the ends of the viral genome.For amplification of related molecules (e.g., mutant forms of the samevirus present in a patient sample), the primers are preferablycomplementary to conserved regions of the target nucleic acid likely tobe present in most members of the population. PCR amplification isdescribed in PCR Technology: Principles and Applications for DNAAmplification (ed. H. A. Erlich, Freeman Press, NY, NY, 1992); PCRProtocols: A Guide to Methods and Applications (eds. Innis, et al.,Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic AcidsRes. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17(1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Pat.No. 4,683,202.

3. Detection

Detection of a nucleic acid analyte can be performed following captureand either during (real-time) or following (end-point) amplification byusing any known method. The amplification product of RNA is often in theform of DNA resulting from RT-PCR or RNA copies resulting from TMA.Amplified nucleic acids may be detected in solution phase or byconcentrating them in or on a matrix and detecting labels associatedwith them (e.g., an intercalating agent such as ethidium bromide). Somedetection methods use probes complementary to a sequence in theamplified product and detect the presence of the probe:product complex,or use a complex of probes to amplify the signal detected from amplifiedproducts (e.g., U.S. Pat. Nos. 5,424,413, 5,451,503 and 5,849,481).Other detection methods use a probe in which signal production is linkedto the presence of the target sequence because a change in signalresults only when the labeled probe binds to amplified product, such asin a molecular beacon, molecular torch, or hybridization switch probe(e.g., U.S. Pat. Nos. 5,118,801, 5,210,015, 5,312,728, 5,538,848,5,541,308, 5,656,207, 5,658,737, 5,925,517, 6,150,097, 6,361,945,6,534,274, 6,835,542, and 6,849,412; and U.S. Pub. No. 2006/0194240 A1).Such probes typically use a label (e.g., fluorophore) attached to oneend of the probe and an interacting compound (e.g., quencher) attachedto another location of the probe to inhibit signal production from thelabel when the probe is in one conformation (“closed”) that indicates itis not hybridized to amplified product, but a detectable signal isproduced when the probe is hybridized to the amplified product whichchanges its conformation (to “open”). Detection of a signal fromdirectly or indirectly labeled probes that specifically associate withthe amplified product indicates the presence of the target nucleic acidthat was amplified.

4. Sequencing

Following amplification, a nucleic acid analyte as well as or instead ofundergoing qualitative or quantitative detection can be sequenced.Purification if desired can be performed on a silica column (e.g., aQiagen gravity flow column). The target nucleic acid binds to thecolumn, where it can be washed and then eluted. Alternatively,purification can be performed using a nucleic acid probe-basedpurification system (e.g., U.S. Pat. No. 6,110,678 or 8,034,554, US2013/0209992 or US 2009/0286249, or WO 2012/037531 or WO 2013/116774).The amplified analyte DNA can also be adapted for some sequencingformats by attachment of an adapter. The amplified DNA can be tailed byKlenow-mediated addition of nucleotides (usually a homopolymer) followedby annealing to an oligonucleotide complementary to the added tail, andligation. Depending on the sequencing platform used, special adaptorsare ligated to the template before sequencing. For example, a SMRT belladapter is ligated to the sample template for sequencing with a PacificBiosciences' PacBio RS sequencer (see, e.g., Travers et al. Nucl. AcidsRes. (2010) 38 (15): e159).

The amplified target nucleic acid is suitable for sequence analysis by avariety of techniques. The capture of target nucleic acid can be coupledto several different formats of so-called next generation and thirdgeneration sequencing methods. Such methods can sequence millions oftarget templates in parallel. Such methods are particularly useful whenthe target nucleic acid is a heterogeneous mixture of variants. Amongthe many advantages, sequencing variants in parallel provides a profileof drug resistant mutations in the sample, even drug mutations presentin relatively minor proportions within the sample.

Some next generation sequence methods amplify by emulsion PCR. A targetnucleic acid immobilized to beads via a target capture oligomer providesa suitable starting material for emulsion PCR. The beads are mixed withPCR reagents and emulsion oil to create individual micro reactorscontaining single beads (Margulies et al., Nature 437, 376-80 (2005)).The emulsion is then broken and the individual beads with amplified DNAare sequenced. The sequencing can be pyrosequencing performed forexample using a Roche 454 GS FLX sequencer (454 Life Sciences, Branford,Conn. 06405). Alternatively, sequencing can be ligation/detectionperformed for example using an ABI SOLiD Sequencing System (LifeTechnologies, Carlsbad, Calif. 92008). In another variation, analytenucleic acids are eluted from beads having target capture oligomers andare immobilized in different locations on an array (e.g., the HiScanSQ(Illumina, San Diego, Calif. 92121)). The target nucleic acids areamplified by bridge amplification and sequenced by template directedincorporation of labeled nucleotides, in an array format (Illumina). Inanother approach, analyte nucleic acids are eluted from the targetcapture oligomer and single molecules are analyzed by detecting inreal-time the incorporation nucleotides by a polymerase (single moleculereal time sequencing or SMRT sequencing). The nucleotides can be labelednucleotides that release a signal when incorporated (e.g., PacificBiosciences, Eid et al., Sciences 323 pp. 133-138 (2009) or unlabelednucleotides, wherein the system measures a chemical change onincorporation (e.g., Ion Torrent Personal Genome Machine (LifeTechnologies)).

Although captured target nucleic acids can be sequenced by anytechnique, third generation, next generation or massively parallelmethods offer considerable advantages over Sanger and Maxam Gilbertsequencing. Several groups have described an ultrahigh-throughput DNAsequencing procedure (see. e.g., Cheeseman, U.S. Pat. No. 5,302,509,Metzker et al., Nucleic Acids Res. 22: 4259 (1994)). The pyrosequencingapproach that employs four natural nucleotides (comprising a base ofadenine (A), cytosine (C), guanine (G), or thymine (T)) and severalother enzymes for sequencing DNA by synthesis is now widely used formutation detection (Ronaghi, Science 281, 363 (1998); Binladin et al.,PLoS ONE, issue 2, e197 (February 2007); Rehman et al., American Journalof Human Genetics, 86, 378 (March 2010); Lind et al., Next GenerationSequencing: The solution for high-resolution, unambiguous humanleukocyte antigen typing, Hum. Immunol. (2010), doi10.1016/jhumimm.2010.06.016 (in press); Shafer et al., J Infect Dis.1;199(5):610 (2009)). In this approach, the detection is based on thepyrophosphate (PPi) released during the DNA polymerase reaction, thequantitative conversion of pyrophosphate to adenosine triphosphate (ATP)by sulfurylase, and the subsequent production of visible light byfirefly luciferase. More recent work performs DNA sequencing by asynthesis method mostly focused on a photocleavable chemical moiety thatis linked to a fluorescent dye to cap the 3′—OH group of deoxynucleosidetriphosphates (dNTPs) (Welch et al. Nucleosides and Nucleotides 18, 197(1999) & European Journal, 5:951-960 (1999); Xu et al., U.S. Pat. No.7,777,013; Williams et al., U.S. Pat. No. 7,645,596; Kao et al, U.S.Pat. No. 6,399,335; Nelson et al., U.S. Pat. Nos. 7,052,839 & 7,033,762;Kumar et al., U.S. Pat. No. 7,041,812; Sood et al, US Pat. App. No.2004-0152119; Eid et al., Science 323, 133 (2009)). Insequencing-by-synthesis methodology, DNA sequences are being deduced bymeasuring pyrophosphate release on testing DNA/polymerase complexes witheach deoxyribonucleotide triphosphate (dNTP) separately andsequentially. See Ronaghi et al., Science 281: 363 365 (1998); Hyman,Anal. Biochem. 174, 423 (1988); Harris, U.S. Pat. No. 7,767,400.

B. Other Analytes

Antibodies, proteins, particles and other analytes can be detected byformats such as immunoprecipitation, Western blotting, ELISA,radioimmunoassay, competitive and immunometric assays. See Harlow &Lane, Antibodies: A Laboratory Manual (CSHP NY, 1988); U.S. Pat. Nos.3,791,932; 3,839,153; 3,850,752; 3,879,262; 4,034,074, 3,791,932;3,817,837; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517;3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and4,098,876. Sandwich assays are a preferred format (see U.S. Pat. Nos.4,376,110, 4,486,530, 5,914,241, and 5,965,375).

Competitive assays can also be used. In some methods, analyte antigen ina sample competes with exogenously supplied labeled analyte antigen forbinding to an antibody detection reagent. The amount of labeled analyteantigen bound to the antibody is inversely proportional to the amount ofanalyte antigen in the sample. The antibody can be immobilized tofacilitate separation of the bound complex from the sample prior todetection.

Lateral flow devices can also be used for detecting an analyte. Fluid isapplied to a test strip that has been treated with a sample in which ananalyte may be present. Labelled binding molecules pass through thestrip and can be captured as they pass into a specific zone containingthe sample with the analyte.

VI. Sensitivity

The present methods can provide a high sensitivity of detection of ananalyte from blood cells. For pathogen-derived RNA analytes, sensitivitycan be expressed as a minimum number of pathogenic RNA copies present ina volume of whole blood. The volume of whole blood can be that contactedwith lysis reagent directly, or can be that used to prepare a bloodfraction, such as pelleted red cells, which are in turn contacted withthe lysis reagent. Preferably the methods detect the presence ofpathogenic RNA in whole blood with a sensitivity of about 2×10³ copiesof ribosomal RNA/mL (equivalent to one parasite/1 mL) of whole blood orbetter, 2×10³ copies/5 mL of whole blood or better, 2×10³ copies/10 mLof whole blood or better, 2×10³ copies/50 mL of whole blood or better,or 2×10³ copies/100 mL of whole blood or better. Preferably the methodsdetect the presence of pathogenic RNA in whole blood with a sensitivityof about 8×10³ copies of ribosomal RNA/mL (equivalent to fourparasites/1 mL) of whole blood or better, 8×10³ copies/5 mL of wholeblood or better, 8×10³ copies/10 mL of whole blood or better, 8×10³copies/50 mL of whole blood or better, or 8×10³ copies/100 mL of wholeblood or better. Preferably the methods detect the presence ofpathogenic RNA in whole blood with a sensitivity of about 24×10³ copiesof ribosomal RNA/mL (equivalent to 12 parasites/1 mL) of whole blood orbetter, 24×10³ copies/5 mL of whole blood or better, 24×10³ copies/10 mLof whole blood or better, 24×10³ copies/50 mL of whole blood or better,or 24×10³ copies/100 mL of whole blood or better.

EXAMPLES Example 1. Analysis of Reagents for Cell Lysis andStabilization of Babesia RNA

The purpose of this example was to identify a lysis reagent that wouldeffectively and preferentially lyse red blood cells in human wholeblood, stabilize analyte(s) in the lysed sample, and inhibit theactivity of RNAses. Preferential lysis of red blood cells over othercellular components of blood means that the percentage of red bloodcells lysed is higher than that of other cellular components present inthe sample being analyzed, the other cell types being assessed in theaggregate. In this example, the analyte is a pathogen-derived RNAanalyte, 18S ribosomal RNA from Babesia parasites. To be compatible withGen-Probe's Target Capture Technology using magnetic beads, the lysisreagent should preferably result in a homogeneous lysate for efficienttarget capture.

In this first example, the PAXgene™ Blood RNA System (BD Biosciences),Lysis Reagent A and Lysis Reagent B were evaluated for Babesia samplepreparation. The PAXgene reagent contained in each tube comprises theactive compound tetradecyltrimethylammonium oxalate (TDTMAO), aquarternary ammounium salt known to lyse cell membranes and act as astabilizing reagent. Lysis Reagent A, was an aqueous solution of 14 mMsodium bicarbonate, 250 mM ammonium chloride, 5% (w/v) LLS, and 0.1 mMEDTA, at a pH of 7.4. Lysis Reagent B, was an aqueous solution of 14 mMsodium bicarbonate, 250 mM ammonium chloride, 8% (w/v) LLS, and 0.1 mMEDTA, at a pH of 7.3.

The sample used for preparation was human whole blood spiked withBabesia-infected hamster blood. Infected hamster whole blood wasserially diluted by combining 10 uL of infected hamster blood with 90 uLof fresh human donor blood (uninfected). Each of these serial dilutionswere then combined with 900 uL of fresh human donor blood to provide 1mL samples. Each 1 mL sample was first combined with 3 mL of lysisreagent from a PAXgene tube at room temperature and allowed to rock for5 minutes to induce cell lysis. 500 μL of the lysed sample was thenadded to 500 μL of a Target Capture Reagent (TCR). Gen-Probe, Procleix,and Aptima TCRs were evaluated. Following addition of the lysed sampleto the TCR, a white precipitate formed. Therefore, the PAXgene systemwas unsuitable for whole blood lysis, capture, amplification anddetection of Babesia using Gen-Probe's target capture, amplification anddetection reagents.

In a next experiment, a lysis reagent of 250 mM ammonium chloride (ACL),buffered with 14 mM sodium bicarbonate and containing LLS and EDTA, wasevaluated. Human whole blood was spiked with Babesia-infected hamsterblood at a dilution ranging from 1×10⁻⁵ to 1×10⁻⁸, as generallydescribed above. One mL of spiked whole blood was then admixed with 3 mLof Lysis Reagent A for 5 minutes at 25° C. to induce red blood celllysis. Following the addition of 500 μL of the lysed sample to 500 μLTCR, no precipitate was observed. Target capture was performed asgenerally described in U.S. Pat. No. 6,110,678. Babesia 18S rRNA wasdetected in each sample by transcription-mediated amplification (U.S.Pat. Nos. 5,399,491, 5,554,516, 5,824,518 and 7,833,716). Six (6)replicates of each dilution condition were amplified and detected.Parasite load was determined based on statistically identifying thedilution to contain about 1 parasite per mL. In addition, a serialdilution of an IVT stock with a known concentration was amplified anddetected in separate wells of the reaction in order to provide a curvefor calculating parasite load in each amplified/detected dilution of thehamster blood. Target capture oligomers, primers and probes used tocapture, amplify and detect Babesia 18S rRNA in the samples were asfollows:

TABLE 1 FUNCTION Sequence (5′-3′) non T7 PrimerACAGGGAGGTAGTGACAAG (SEQ ID NO: 1) T7 PrimerAATTTAATACGACTCACTATAGGGAGACTGGAATT ACCGCGGCTGCTGG (SEQ ID NO: 2)AE Probe ACCCUUCC CA GAGUAUCAAU (SEQ ID NO: 3) TCOGGAUUGGGUAAUUUGCGCGCCTTTAAAAAAAAAAA AAAAAAAAAAAAAAAAAAA (SEQ ID NO: 4)

Amplification and detection results from each of the conditionsmentioned above show that Lysis Reagent A effectively lysed red bloodcells to release Babesia 18S rRNA for subsequent analysis. Comparison ofthe Babesia rRNA detected in each of these samples of the serialdilution to the results from the serially diluted IVT showed a limit ofdetection as low as 0.01 parasites per mL.

Additional lysis reagents were evaluated for their lysis of blood cellsand detection of pathogen derived analytes.

Example 2. Evaluation of Additional Blood Cell Lysis Reagents

A study of lysis reagents was performed to evaluate their ability toeffectively lyse blood cells and release analytes for subsequentevaluation. Nucleic acid analytes were evaluated in this example using aTMA amplification and detection reaction to identify 18S rRNA fromBabesia parasite, as described herein.

The sample used in this example was Babesia infected human whole blood,determined to be positive for Babesia by PCR. Parasitemia was determinedby serially diluting the Babesia infected blood into uninfected blood,then increasing the volume of each dilution to 1 mL using uninfectedblood, mixing the 1 mL dilution with 3 mL of Lysis Reagent A, and thenperforming capture, amplification and detection reactions as describedin Example 1, above. Parasite load in the stock infected human bloodsample was determined based on statistically identifying the dilution tocontain about 1 parasite per mL and then back calculating to the stockinfected blood sample. The infected blood sample was then separatelydiluted to provide 12 parasite/mL (12 p/mL) and 4 parasite/mL (4 p/mL)dilutions at a total volume of 1 mL, as generally described. The 12 p/mLand the 4 p/mL were each used in the below assays.

Lysis reagent C was made similarly to Lysis Reagent B, but theconcentration of EDTA was increased to 10 mM. Lysis reagent D was anaqueous solution of 15 mM sodium phosphate monobasic, 15 mM sodiumphosphate dibasic, 10% (w/v) LLS, 1 mM EGTA, and 1 mM EDTA-Nazdihydride, at a pH of 6.7. Lysis reagent E was an aqueous solution of100 mM TRIS 30 mM magnesium chloride, and 6% (w/v) LLS, at pH 7.5.Separate spiked whole blood samples (described above) were each lysedwith one of lysis reagents C-E at a ratio of 1:2 or 1:4 (bloodsample:lysis reagent) and tested at 36 to 72 replicates per condition,as identified in Tables 2-4. Reactive samples were determined to bethose with an RLU value greater than 100,000 RLU. Babesia 18S rRNA wasdetected in each sample using Procleix TCR and amplification by TMA asdescribed above. Conditions were evaluated for sensitivity, stability,and robustness. Results for lysis reagents C-E are shown in Table 2,Table 3, Table 4.

TABLE 2 Sensitivity Lysis Number of Number Percent Ratio ConcentrationReagent Reactions Reactive Reactive 1:2 12 p/mL C 71 70 98.5% D 72 72100.0% E 72 72 100.0%  4 p/mL C 72 50 69.4% D 72 66 91.7% E 69 54 78.3%1:4 12 p/mL C 72 72 100.0% D 72 72 100.0% E 72 72 100.0%  4 p/mL C 72 6691.7% D 72 63 87.5% E 72 67 93.1%

TABLE 3 Stability: One Day. Day 0 Day 1 (stored at 4° C.) Lysis Numberof Number Percent Number of Number Percent Ratio Conc. Reagent ReactionsReactive Reactive Reactions Reactive Reactive 1:2 12 p/mL C 35 35 100.0%36 34  94.4% D 36 36 100.0% 36 35  97.2% E 36 36 100.0% 36 36 100.0% 4p/mL C 36 28  77.8% 36 20  55.6% D 36 31  86.1% 36 30  83.3% E 36 27 75.0% 36 33  91.7% 1:4 12 p/mL C 36 36 100.0% 36 36 100.0% D 36 36100.0% 36 36 100.0% E 36 36 100.0% 36 36 100.0% 4 p/mL C 36 32  88.9% 3635  97.2% D 36 31  86.1% 36 36 100.0% E 36 33  91.7% 36 35  97.2%

TABLE 4 Stability: Three Day. Day 0 Day 3 (stored at 4° C.) Lysis Numberof Number Percent Number of Number Percent Ratio Conc. Reagent ReactionsReactive Reactive Reactions Reactive Reactive 1:2 12 p/mL C 36 35  97.2%36 12  33.3% D 36 36 100.0% 36 36 100.0% E 36 36 100.0% 36 25  69.4% 4p/mL C 36 22  61.1% 36  9  25.0% D 36 35  97.2% 36 27  75.0% E 36 27 81.8% 36 24  66.7% 1:4 12 p/mL C 36 36 100.0% 18 18 100.0% D 36 36100.0% 24 24 100.0% E 36 36 100.0% 24 24 100.0% 4 p/mL C 36 34  94.4% 3631  86.1% D 36 32  88.9% 36 29  80.6% E 36 34  94.4% 36 32  88.9%

These data show that lysis reagents C-E performed well in lysing wholeblood and releasing pathogen-derived analytes from blood cells forsubsequent analysis. The analytical sensitivity of a TMA assay foramplification and detection of Babesia 18S rRNA obtained from bloodcells using lysis reagents C-E was at least as low as 4 p/mL and at adilution of lysis buffer to whole blood as low as 4:1. A loss ofsensitivity was observed following storage at 4° C. after three days, asseen by the large variability in results. This loss in sensitivity wasreadily observable in samples having a 2:1 dilution and 4 p/mL.

Although the invention has been described in detail for purposes ofclarity of understanding, certain modifications may be practiced withinthe scope of the appended claims. All publications including accessionnumbers, websites and the like, and patent documents cited in thisapplication are hereby incorporated by reference in their entirety forall purposes to the same extent as if each were so individually denoted.To the extent difference version of a sequence, website or otherreference may be present at different times, the version associated withthe reference at the effective filing date is meant. The effectivefiling date means the earliest priority date at which the accessionnumber at issue is disclosed. Unless otherwise apparent from the contextany element, embodiment, step, feature or aspect of the invention can beperformed in combination with any other.

What is claimed is:
 1. A blood cell lysis reagent comprising: (i) TRISat a concentration from about 90 mM to about 110 mM, (ii) lithium laurylsulfate (LLS) at a concentration of from about 5% (w/v) to about 8%(w/v), and (iii) magnesium chloride at a concentration of from about 28mM to about 40 mM, wherein the reagent has a pH that is from about 7.0to about 8.0, and wherein the reagent does not include an anti-coagulantselected from the group consisting of EDTA, EDTA-Na₂, EGTA, andcombinations thereof.
 2. The reagent of claim 1, wherein the magnesiumchloride is present at a concentration of from about 28 mM to about 35mM.
 3. The reagent of claim 2, wherein the magnesium chloride is presentat a concentration of about 30 mM.
 4. The reagent of claim 1, whereinthe LLS is present at a concentration of about 6% (w/v).
 5. The reagentof claim 1, wherein the TRIS is present at a concentration of about 100mM.
 6. A composition comprising the reagent of claim 1 and blood cells.7. The composition of claim 6, wherein the composition comprises wholeblood.
 8. A method of lysing blood cells to release an analytetherefrom, comprising the steps of: (a) contacting a sample containingblood cells with a lysis reagent that is effective to lyse the bloodcells and release therefrom an analyte for analysis, wherein the lysisreagent comprises (i) TRIS at a concentration from about 90 mM to about110 mM, (ii) lithium lauryl sulfate (LLS) at a concentration of fromabout 5% (w/v) to about 8% (w/v), and (iii) magnesium chloride at aconcentration of from about 28 mM to about 40 mM, wherein the lysisreagent has a pH that is from about 7.0 to about 8.0, and wherein thelysis reagent does not include an anti-coagulant selected from the groupconsisting of EDTA, EDTA-Na2, EGTA, and combinations thereof; (b)providing conditions for lysing blood cells in the sample whereby atleast a portion of the blood cells are lysed and the analyte releasedtherefrom; and (c) analyzing the analyte released in step (b).
 9. Themethod of claim 8, wherein the analyte is a pathogen-derived analyte.10. The method of claim 8, wherein the analyte is an RNA analyte. 11.The method of claim 8, further comprising the step of immobilizing theanalyte on a solid support.
 12. The method of claim 11, wherein the stepof immobilizing the analyte comprises contacting the released analytewith a capture probe and an immobilized probe, the capture probe havinga first segment complementary to the analyte, and a second segmentcomplementary to the immobilized probe, wherein the analyte binds to thecapture probe, and wherein the bound capture probe binds to theimmobilized probe.
 13. The method of claim 11, wherein the immobilizedanalyte is analyzed using an amplification reaction to amplify theanalyte and detecting the resulting amplification product with adetection probe.
 14. The method of claim 13, wherein the amplificationreaction is an isothermal amplification reaction.
 15. The method ofclaim 8, wherein the method is performed without a centrifugation stepto separate the reagent from the target released from the red bloodcells.
 16. The method of claim 8, wherein the analyte is a pathogenicorganism.
 17. The method of claim 16, wherein the pathogenic organism isselected from the group consisting of: hepatitis viruses, humanimmunodeficiency viruses, dengue viruses, west nile viruses,flaviviruses, zika virus, and parasitic organisms.
 18. The method ofclaim 17, wherein the pathogenic organism is a parasitic organismselected from the group consisting of: parasites from the genus Babesia,parasites from the genus Plasmodium, parasites from the genusTrypanosoma, parasites from the genus Leishmania, parasites from thegenus Anaplasma, parasites from the genus Toxoplasma, Babesia microti,Babesia divergens, Babesia duncani, Plasmodium falciparum, Plasmodiummalariae, Plasmodium ovale, Plasmodium vivax, and Plasmodium knowlesi.19. The method of claim 8, wherein the sample comprises whole blood, andwherein during the contacting step the ratio of reagent to whole bloodis from 1:1 to 4:1.
 20. A method of separating an analyte from a samplecontaining blood cells, consisting essentially of the steps of: (a)incubating a mixture of a lysis reagent and a sample containing bloodcells under conditions sufficient for lysis of at least a portion of theblood cells in the sample, thereby releasing an analyte; (b) contactingthe mixture with a solid support configured to immobilize the analyte;and (c) separating the immobilized analyte from the mixture; wherein thelysis reagent comprises (i) TRIS at a concentration from about 90 mM toabout 110 mM, (ii) lithium lauryl sulfate (LLS) at a concentration offrom about 5% (w/v) to about 8% (w/v), and (iii) magnesium chloride at aconcentration of from about 28 mM to about 40 mM, wherein the reagenthas a pH that is from about 7.0 to about 8.0, and wherein the reagentdoes not include an anti-coagulant selected from the group consisting ofEDTA, EDTA-Na2, EGTA, and combinations thereof.